Project Summary Barth Syndrome (BTHS), is an X-linked inborn error of mitochondrial phospholipid metabolism, caused by variants in the gene tafazzin (TAZ). TAZ encodes for a transacylase involved in the final remodeling step of cardiolipin (CL), a phospholipid localized to the inner mitochondrial membrane with key roles in cristae formation, organization of the mitochondrial respiratory chain, and in the apoptotic cascade. Both TAZ and CL are ubiquitously expressed in all tissues, however the CL content is tissue specific; brain CL is characterized by a diversified array of acyl chains including polyunsaturated chains, whereas in the cardiac and skeletal muscle CL is predominantly characterized by the tetralinoleoyl form. As is typical of most primary mitochondrial diseases, BTHS is a multisystem disorder, characterized by cardiomyopathy, skeletal myopathy, and neutropenia among other features. However, in sharp contrast to many mitochondrial diseases, BTHS has minimal neurological burden. Thus, understanding the mechanisms of tissue specificity in BTHS has the potential to provide great insight into the role of specific CL content on mitochondrial function, as well as to offer novel treatment targets for BTHS. In my preliminary work with a novel TAZ deficient cellular model, I found that complex I of the respiratory chain is dysregulated at the protein and metabolic level. Based on this, I hypothesize that complex I is a major site of oxidative phosphorylation (OXPHOS) dysfunction in tissues affected in BTHS. Dysregulation of complex I in BTHS is a particularly interesting finding, because other complex I disorders, including Leigh Disease, have a significant neurological impact. Therefore, I further hypothesize that the tissue specific CL content of neurologic tissue spares it from this dysfunction. To address this hypothesis, I propose two specific aims. Aim 1: To determine the impact of TAZ deficiency, and therefore abnormal CL content, on complex I function. I will first investigate the effect of the reduced expression of Complex I proteins on enzyme stability and function. Then I will determine whether the assembly factor NDUFAF1 plays a prominent role in the reduced expression of complex I proteins, and if the reduced NDUFAF1 expression is due to dysregulation at the transcriptional level. Aim 2: To understand the role of tissue specificity of CL content on the pathophysiology of BTHS. I will first differentiate TAZ deficient iPSCs, iPSC TAZ?50, into cardiomyocytes and neurons in order to characterize and compare complex I dysregulation by assessing NDUFAF1 protein expression, NADH/NAD+ ratio, and complex I enzyme stability/function. Then, I will determine if TAZ deficient neurons display altered CL as compared to WT neurons, and if the lack of altered CL content in TAZ deficient neurons reflects a smaller role for TAZ in neuronal CL remodeling. This project proposal combines basic science and translational approaches to unravel cellular pathophysiology and identify potential therapeutic targets for a rare disease. The skills I will acquire as I complete this research will prepare me for a career in therapeutic discovery for rare genetic conditions.